LOGAN, UT – “Sometimes an answer is staring you right in the face, but you don’t see it because you’re blinded by a preconceived notion,” says Carol von Dohlen, assistant professor of biology at Utah State University. “Sometimes you have to think outside the box and listen to fresh viewpoints.”

The self-described “insect evolutionary biologist” recently published the discovery she, undergraduate colleagues, Shawn Kohler and Skylar Alsop, and Electron Microscopy Laboratory supervisor William McManus, made of one bacterium living symbiotically within another in the July 26 issue of “Nature.”

“To our knowledge, this is the first documented report of intracellular symbiosis involving two species of bacteria,” said von Dohlen.

The Utah State group’s research involved citrus mealybugs, close cousins of aphids, which feed on sweet plant sap. Scientists have been aware that such insects hosted bacteria since the early 1900s, explains von Dohlen. “The sap is high in sugar, but low in protein. The bacteria in aphids produce amino acids – protein the insects need for survival.”

In aphids and other insects, she says, the bacteria are housed inside special host cells. “But one odd aspect of mealybugs is that the bacteria are packaged in ‘symbiotic spheres’ – of unknown origin – within the host cells.”

While von Dohlen knew that mealybugs housed two types of bacteria, identified as beta-Proteobacteria and gamma-Proteobacteria, she assumed they lived side-by-side or in separate cells. She and her research group set out to examine the two bacterial species using a technique called fluorescent in situ hybridization or FISH.

“I gave Shawn Kohler the assignment of working out the FISH technique and devising probes to identify the bacteria,” said von Dohlen. “Each probe was constructed with a label that would fluoresce at a certain wavelength.”

Using laser-scanning confocal microscopy, the researchers first examined the mealybug tissue using the gamma probe. “We saw beautiful, gorgeous signal from the gamma-Proteobacteria inside symbiotic spheres.”

Using the beta probe, the researchers detected fluorescence inside the same spheres. “We thought, ‘This can’t be. There’s no second form of bacteria in the spheres,’” said von Dohlen. “We were tearing our hair out and Bill (McManus), our electron microscopy supervisor, suggested that the spheres themselves might be beta-Proteobacteria.”

When the group considered McManus’ suggestion, said von Dohlen, “all the data suddenly made sense.”

Further examination with electron microscopy confirmed that the gamma-bacterial endosymbionts did indeed live inside the beta-bacterial symbionts. von Dohlen says the function of the gamma symbionts isn’t known, but she hopes her findings will entice microbiologists to find out. She speculates that the betas may be using gene products from, or even exchanging genetic material with, the gammas.

“The gammas may be providing new material to compensate for genetic defects in the betas,” says von Dohlen.

Bacteriologist Paul Baumann at University of California, Davis calls the Utah State group’s discovery “a major new contribution … one of the most exciting findings in years.”